RECOVERY OF PUTATIVE ISOVANILLIN AND COUMARIN-TYPE AROMATICS FROM COCOA POD HUSK BIOCONVERTED BY Serpula lacrymans: A RESPONSE-SURFACE-OPTIMIZED SOLVENT EXTRACTION

Abstract

Aromatic compounds derived from lignin and pharmaceutical substances are pivotal components in the development of next- generation biorefineries. However, their recovery from tropical agro-residues is infrequently optimized through an integrated approach. Cocoa pod husk (CPH), produced in Indonesia at a rate exceeding 0. 5 million tonnes annually, represents a notably underutilized lignocellulosic feedstock. In this study, we combined a 35- day solid- state pretreatment of CPH with the brown- rot fungus Serpula lacrymans and an ethyl acetate extraction method systematically optimized via Response Surface Methodology (RSM). The pretreatment process was monitored through measurements of total reducing sugar (TRS), total soluble phenol (TSP), pH, and dry- weight loss; the TSP reached a maximum of 0. 304 mg g⁻¹ on day 21, and this sample was selected for subsequent extraction. Utilizing a rotatable central- composite design (CCD) evaluating solvent volume (11. 72–68. 28 mL per 10 g) and extraction time (35. 16–204. 84 minutes), quadratic models with R ² values of 0. 979 (vanillin- equivalent) and 0. 942 (TSP) were established. Significance testing revealed solvent volume as highly influential for both responses (p < 0. 0001), while extraction time was not statistically significant (p > 0. 37). The numerical optimization point (60 mL solvent volume and 96. 96.4 minutes extraction time; desirability = 0. 967) yielded 1. 64% (w/w) of vanillin- equivalent substances and 0. 05 mg g⁻¹ TSP. High- performance liquid chromatography- electrospray ionization- tandem mass spectrometry (HPLC- ESI- MS/MS) analysis of the optimized extract did not detect free vanillin but identified two distinct aromatic peaks, tentatively characterized as isovanillin ([M + H]⁺ = 153, RT = 1. 35 minutes) and a coumarin- like compound ([M + H]⁺ = 147, RT = 3. 93 minutes). Both compounds are potentially bioactive, valuable molecules if their identities are confirmed. Thus, CPH bioconverted by S. lacrymans serves as a source of a structurally diverse aromatic fraction rather than solely vanillin, repositioning this tropical residue as a versatile feedstock for multi- product biorefining rather than vanillin- only extraction.